The Technique
Critical point drying is a method of drying tissue without collapsing or
deforming the structure of wet, fragile specimens, generally as part of
the sample preparation process for scanning electron microscopy (SEM).
Although it has some applications in transmission electron microscopy
(TEM), its major application, at least up until now, has been in tissue
preparation for SEM. It is well known that allowing tissue to dry in air
or under vacuum (during the metallization process, for example) causes
damage to the surface which one wishes to examine in the SEM. An example
is shown in the micrographs shown below:
SEM image (850X) of rose petal surface, CPD
SEM image (850X) of rose petal surface, fixed and air dried
In some cases, this deformation is acceptable during routine experiments,
but for the most part, it is not. At one time, workers in the field tried
to "turn the other way" and pretend these drying artifacts did not exist.
But today's workers, pretty much worldwide, with their new high resolution
SEMs, will not tolerate drying artifacts. And therefore, there is a
universal acceptance of the need for a CPD unit is virtually all life
science laboratories and even in some circumstances, depending on the
nature of the samples being examined, a growing number of materials
science laboratories.
Explanation of drying artifacts
The reason why tissue samples became damaged by normal air drying is that
very large surface tension forces are created in cavities of small
dimensions when there is a liquid/gas interface. As tissue dries, the
liquid/gas interface travels through the surface of the material
collapsing the cavities between projecting structures. In the case of
delicate liquid- containing samples, which become hollow when dried,
complete collapse often results. The critical point drying method of
drying avoids these effects by never allowing a liquid/gas interface to
develop; in this way the tissue is not exposed to surface tension forces.
The critical point:
The critical point of a liquid/gas system (e.g. water/steam, liquid
CO2/CO2gas) is its critical temperature and the
pressure associated with this temperature, that is, it is a point
Tc, Pc smaller on the T,P phase diagram. Above
the critical temperature, Tc the system is always gaseous and
cannot be liquefied by the application of pressure. The transition from
liquid to gas at the critical point takes place without an interface
because the densities of liquid and gas are equal at this point.
If tissue is totally immersed in a liquid below its critical point
and the liquid is then taken to a temperature and pressure above
the critical point it is then immersed in gas (i. e. dried) without being
exposed to the damaging surface tension forces.
In order to carry out this procedure at a convenient temperature and
pressure, it is normal to replace the water (which has a very high critical
point) with some other liquid before carrying out the drying. More than
one substitution is usually necessary if the final liquid is not miscible
with water, e.g. if the final liquid is liquid carbon dioxide, the water
in the tissue is first replaced with acetone and then the acetone is
replaced with liquid CO2.
Another "route" is water-ethanol-Freon® 113-CO2. However
because of environmental concerns, and the general unavailability any
longer of Freon 113, any route depending on Freon 113 is generally not any
longer practiced. Liquid CO2 is the most usual drying medium
because it is inexpensive, convenient, and environmentally acceptable, at
least relative to Freon 113. Nitrous oxide has also been used for this
purpose but it has never achieved much acceptance.
Fixation with glutaraldehyde and
osmium tetroxide followed
by the substitution of acetone or Freon 113 is carried out before
transferring the tissue to the
critical point drying
apparatus. Final substitution with liquid CO2 and the drying
run are carried out inside the apparatus. After the drying run, the
pressure is released and the dried tissue can then be metallized before
being inserted into the SEM for observation and imaging. Usually, the
metal used is gold and this is done in a
sputter coater
or osmium coater.
The comparison micrographs shown above shows an example of tissue which has
been fixed in gluteraldehyde and osmium tetroxide, substituted with ethanol
and then Freon 113, and finally critical point dried from liquid CO2.
The difference between the two micrographs shows the obvious advantages of
using the technique of critical point drying on these kinds of samples.
The Apparatus Itself
The main body of the apparatus is a pressure vessel with integral water
jacket for heating and cooling. The normal operating range of the pressure
chamber is 0-2000 psi and 10-50°C. As can be seen in the
photo of the unit, one can
see various control valves, a thermometer, a pressure gauge and a support
stand are all attached to the vessel. At one end of the cylindrical
chamber is a demountable window for viewing the process and in the
opposite end a removable access door for the specimen holder. The viewing
window is an indispensable part of the design of the SPI CPD unit because
it is so important to make sure that there is no turbulence when the
CO2 is being run through the unit.
There are four pressure control valves. Built into the support column is an
over-pressure safety valve, sometimes called also a rupture disc, set at
2000 psi. Should this pressure be exceeded by overheating the chamber,
the value opens and reduces the pressure to ambient. This rupture disc
must be replaced before the unit can be used further.
The manual value at the top rear of the body is used for admitting the
liquid gas to the chamber. A transfer pipe with couplings is provided
for connecting the apparatus to a suitable siphon cylinder. The manual
valve at the top front of the body is used for venting trapped air when
filling the chamber. The valve at the bottom rear of the body is used
for draining transfer fluid after filling with liquid gas.
Both the vent and drain valves are used for causing a thorough mixing
action when substituting the transfer fluid with the liquid gas. It is
important that all transfer fluid be removed from the tissue and flushed
from the chamber if efficient drying is to be carried out. Turbulence
and flushing are achieved by opening the inlet and drain (or vent) valves
simultaneously.
Care must be taken to ensure that the tissue remains below the level of the
liquid during this operation. And one must avoid turbulence if their samples
are especially fragile in order to avoid artifacts and other damage.
The tissue holder consists of a boat shaped liquid holder in which are
placed tissue baskets with lids. There is an automatic drain in the boat
which acts when the access door is closed with the holder inside the
chamber. This design fulfills two requirements: (a) that the tissue
remains wet during transfer to the apparatus and (b) that the transfer
liquid can be totally removed before the drying run is started.
After replacing the transfer liquid (e.g. acetone, amyl acetate, Freon 113)
with the critical point drying liquid (e.g. liquid CO2) the
drying run can be started. All the valves are closed and hot water is
circulated through the water jacket. In the case of liquid CO2
raising the chamber temperature to 32°C causes a pressure rise from
about 800 psi to about 1150 psi. At this point, the liquid/gas meniscus
becomes diffuse and then disappears. The chamber now contains only gas.
The vent valve can be opened slightly and the gas bled off to leave dry
tissue. To ensure that recondensation of the liquid does not occur, we
recommend that the temperature should be taken at least 5 C° above the
critical point.
Safety precautions
Should any pressure leaks develop during the use of the apparatus, these
are easily fixed as nearly all the seals are standard nitrile rubber
O-rings or bonded seals. For use with aggressive solvents and acetone,
we recommend the use of special EPDM bonded seals. They cost a bit more
but they can be expected to last longer as well.
To Ask a Question or Make a Comment